Fractionation of plant pigments



June 13, 1950 R, MILLER' 2,511,824

FRACTIONATION OF PLANT PIGMENTS Filed April 29, 1947 /JTTORNY Patented June 13, 1950 STATES P'I'IELNT"r OFFICE" i f 2,511,82rf

FRACTIONATION 0F PLANT PIG-MENTS Ralph Miller, Woodside, N'.Y Yi, assigner -to "The Chemical Foundation, Incorporated, a memv bershipy corporation of N evv- York Application April'29, 1947.,`Sex;ial No. 744,195.

14 Claims. l.4

This invention isv 'concernedwith the-'fractionation of plant pigments; with'special" emphasis ony the separationl of carotene `from'chlorophyll.A

. Itis known that .plantslive and' grow through the biological process known. as photosynthesis. This. process, is intimately associated with chlorophyll,v v.a green materialof complex structure found in., all living 'green plants. invariably, this plant' pigment is accompaniedl by other plant pigments including carotene and xanthophylL Carotene can be converted into vitamin A in the body Vof man and many other animals: and hence isof, importance in humanaud animal nutrition. Carotene, chlorophyll and other plant pigments contained in plants are not markedly stable under ordinary conditions so that they tend to be destroyed if the, plants' in'which they'are contanedjare stored forv anylength of time. For certain Iuses, concentrates. of carotene and chlorophyll are required` which necessitate their isolation from the plant material `in whichthey are f ound-` For these` reasons plantscontaining these constitilents aretreated' to remove and recover them ina relatively pure, and concentrated form.

Ordnarily,l plants from which pigments` are L extracte'dare dried and 'ground-prior. to being contacted with a solvent for the pigments. Manyv types of. solvents' can :be used, such as hydrocarbonsa` lgetrones,y chlorinated hydrocarbons and the like.,

,In Yaddition to the Substances mentioned above, plants contain other 'constituentswhich are soluble .in these `solventsysuch as lipids. The solvent and plantmateral are ncontacted under condi-` tions favorable'to the solution of the extractable constituents' in the solvent. The solvent solution is then separatedv from the insoluble residue bysettlin'g, iiltrationggcentrifugingy or any other suitable means.. The lextract solution is then usuallyconcentrated 'by removing part or all of the. solvent'by'distillation. This procedure resuits in 'the' 'production of a crude concentrate whichfrequires additional treatment to produce marketable.. carotene concentrates and marketable chlorophyll-containing mixtures. Crude concentrates suchjas these are complex because they are made'upof'the total fat soluble constituents...of, a-livng organism and :even simple living organisms. centaine large., number of compounds` inthscla'ss. Ally physical method `of separatingA them .iscomplicated `because the .action of the separating agent, on any particular constituent oflthemixlture is affected by somev or all of the other ,constituents inl the mixture. This meansv thatto. obtain a clear Cuneicient (from the yield standp.oint)' separation of' 4any. two constituents ofthemixtnre it is advantageous that each fractionfbe .subjectsdl 'tothe separatin-g'agency repeatedly.

The fact that extracts' from"1iving organisms (ci. zeon-314)@ n such as those from plant tissue are mixtures of many compounds each'of' which influences the behavior of the other constituents, has prevented the'successful development of any simple method of separating chlorophyll from carotene using selective solvents although much work has been carrieda out in an effort to accomplish this objective;

Substantially all proposed methods of separatingcarotene;from'chlorophyll have used either saponication or adsorption or a combination of these two unit processes. These processes are time consuming andv expensive. They require the consumption'of reagents andare ill-adapted to continuousoperation. Consequently, they involvef-constanty supervision of well trained pers'onnel.V Infaddition; they tend to destroy some of-`th"e Vuseful constituents found in the plant extracts:

Itf is a principal objective of this invention to separate'- mixtures containing carotene and chlorophyll into fractions in which each of these constituents 'are concentrated without vconsuminganyreagents and under conditions which precludefany vappreciable decomposition of these substances.A

A second objective of this invention is to producea'caroteneconcentrate which possesses the typical color of a carotene preparation, not brown 'orfblack, and ltoaccomplish this without consumingwany reagents.

A third vobjective-ofl this' invention is the production l'of `a carotene concentrate' of enhanced stability-lcompared 'with' carotene concentrates y prepared-by methods known previously A fourthlobjective: of this'in'vention is' to provide'an economical-method by which the repetitive'treatment'mentioned above of each'phase containing either the carotene or the chlorophyll can` be accomplished:

Another advantage of this invention is that it e'c'tivelyseparates the fatty acids and fats in thef crude concentrate from the' chlorophyll. The oost' ot separatingvchlorophyll from carotene by' direct" adsorption is tcofhigh' tobe employed on an industrialfvscale. Consequently, saponication is practically' universallyemployed'.` When the plant extract-istreated by alk`ali,not'only isthe chloroph-yllisaponie'd but thefat and fatty acids are saponied aswell. Thevcarotene is unaffected which"permitsl-the Vcarotenefto beseparated from' t-hefsaponiedconstituents As a. `result of this operation-the chlorophyll inthe saponied formk is ymixed withrthe Asoapsiformed from the fats f f andiatty acidss'. Thiscomplicates the recovery of chlorophyll or chlorophyll 'derivatives from the mixture of saponiedrmaterials." In the presentl process-the chlorophyllis `not only separatedfromr the carotenewbut it'lisalso lseparated fromthev lipids, thussimplifyingthe recovery and refining'- of chlorophyll and its derivatives. In order to more clearly explain the invention, the process is illustrated in flow sheet form in the accompanying drawing.

It is known that the solubility of pure carotene in paraffin hydrocarbons at relatively low temperatures is small. Similarly, the solubility of pure chlorophyll in paraffin hydrocarbons is very low. However, in the presence of each other and the large amount of lipoidal matter in plant tissue, paraffin hydrocarbons readily dissolve chlorophyll and carotene along with the other fat soluble constituents of plant tissue. In fact, as stated above, paraffin hydrocarbons are employed to extract the fat soluble constituents from plants.

Although paraffin `hydrocarbons can be used to produce crude chlorophyll-carotene-containing concentrates, I have discovered that liquefied, normally gaseous, paraflinic hydrocarbons such as propane, butane, isobut/ane or suitable mixtures of ethane and propane and mixtures of similar solvent characteristics, permit such crude concentrates to be separated into fractions one of which is more concentrated with respect to chlorophyll than the original mixture and the other is more concentrated with respect to carotene than the original mixture.

The preferred solvent is propane. When a crude chlorophyll-carotene containing concentrate is treated with suflicient liquefied propane at a temperature of about 160 F. and a pressure sufficiently high to maintain the propane in the liquid state, two imm'iscible, liquid phases are formed. The lighter phase is composed primarily of propane plus some vof the lipoidal matter; the denser lphase contains most of the black and brown color bodies, substantially al1 the chlorophyll and the remainder of the lipoidal matter. Some of the carotene is in the propane phase and some in the chlorophyll phase. The two phases are easily separated. After this is done, the propane in the lighter phase is readily removed and recovered by distillation. The residue consists of the lipoidal matter originally in the lighter phase plus the carotene present in that phase. It is lighter in color than the charge material and substantially free from chlorophyll. The denser phase containing practically all the chlorophyll and the remainder of the lipoidal matter and carotene may be treated again in the same way with another portion of propane to recover an additional quantity of chlorophyll-free carotene. This procedure can be repeated-until practically all the carotene is freed from chlorophyll. Such amethod of procedure, while feasible, is neither as efficient nor as practical as a continuous method of treating crude concentrate with liquefied, normally gaseous hydrocarbons or mixtures thereof. The preferred method of effecting this separation by means of liquefied, normally gascous hydrocarbons as exemplified by propane, is to use a continuous liquid-liquid extraction system suitable for use with liquid propane at elevated temperatures and pressures. A tower adapted for liquid-liquid extraction under the specified conditions and equipped with means for heating the tower contents at various locations is the preferred type of contacting equipment. The mixture containing the carotene and chlorophyll is pumped into the tower close to its top. Liquid propane is pumped into the tower at a lower level. The temperature is maintained above about 160 F. at the top of the tower. A somewhat lower temperature may prevail near the base of the tower. The mixture of chlorophyll and carotene flows down the tower and meets a rising stream of liquefied hydrocarbon. 'Under the conditions that exist in the tower, two immiscible liquid phases are present. The propane phase is less dense than the chlorophyll containing phase and therefore it rises while the chlorophyll containing phase flows downward. As the two phases flow past each other they are brought into intimate contact by means of the packing or baffles in the tower. The carotene under the conditions existing in the tower passes from the chlorophyll containing phase to the propane phase but under the same conditions substantially none of the clorophyll will dissolve in the propane phase. Most of the lipids present in the feed to the tower also pass into the propane phase. The propane phase under the conditions prevailing in the tower does not dissolve any appreciable amount of the brown or black colored bodies in the feed. A suitable ratio of selective solvent to crude concentrate depends upon the type of plant from which the concentrate was prepared, the prior history of the plant and similar considerations. In general, from 10 to 40 parts by weight or more of solvent to crude concentrate are used.

The propane phase leaves the tower at its top and is then pumped to solvent recovery equipment in which the propane is removed, recovered, and recycled to the extraction operation. The propane free residue is usually dark red in color due to its carotene content. If any free fatty acids are present in the crude concentrate, they will accompany the carotene. It is convenient to remove them from the propane solution by continuously contacting the propane solution flowing from the top of the extraction tower with alkali to react with the fatty acids to form a soap insoluble in the propane solution and continuously separating the soap from the propane solution before the propane solution flows into the solvent recovery equipment.

It will be appreciated that this method of separating the fatty acids involves essentially simple neutralization eiected in the absence of oxygen and in a relatively brief period. Hence, this type of treatment differs materially from the usual method of saponication which involves the relatively long period of contact between the alkali and triglycerides to form a soap and glycerme.

The chlorophyll-containing phase flows out of the bottom of the extraction tower. A small amount of solvent accompanies it. This solvent is removed, recovered and recycled in the conventional manner. The residue contains the chlorophyll, the brown and black color bodies and any solid extraneous substances which were present in the crude concentrate. The values contained in the chlorophyll concentrate may be worked up by known methods or may be used as such where the dark color is not objectionable.

It is known that tocopherols are antioxidants for carotene preparations and that they are usually present in the lipoidal matter of green plants. The tocopherols present in the crude concentrate charged to the extraction tower accompany the carotene into the propane phase and are substantially completely recovered in the carotene concentrate. As a result of this discovery that both the carotene and the tocopherols accompany the propane phase while the chlorophyll remains substantially insoluble in it under the conditions existing in the tower, it is. possible to produce other substantially all of the chlorophyll in the said extract which comprises, contacting such extract with a liquefied, normally gaseous paraiinic hydrocarbon in a continuous liquid-liquid extraction system at a pressure suiciently high to maintain the hydrocarbon in liquid phase and at a temperature sufficiently elevated to insure the formation of two immiscible liquid phases of different densities; withdrawing from the system the denser liquid phase containing substantially all of the chlorophyll and the less dense liquid phase containing substantially all of the carotene, lipids, tocopherols and fatty acids and most of the hydrocarbon, contacting such withdrawn less dense liquid phase with an alkali to convert thefatty acids to soaps insoluble in the hydrocarbon, separating the soaps, and removing the hydrocarbon to recover a concentrate enriched in carotene, lipids and tocopherols.

6. The process of separating an extract of plant tissue containing carotene and chlorophyll into two fractions one of which contains substantially all the carotene and the other substantially all the chlorophyll which comprises, continuously passing said extract into a continuous countercurrent extraction system close to one end of said system and continuously passing propane into said continuous counter-current extraction system close to the other end, at a pressure sufiiiciently high to maintain the propane in liquid phase and at a temperature sufliciently elevated to insure the formation of two immiscible, separable, liquid phases; continuously withdrawing from the said system one liquid phase containing substantially all of the carotene and most of the propane; and withdrawing from the said system the other liquid phase containing substantially all the chlorophyll and the remainder of the propane.

'7. A process of fractionating a crude chlorophyll-carotene-containing mixture into fractions one of which has a greater ratio of carotene to chlorophyll than the original mixture and the other a ratio of chlorophyll to carotene that is greater than that in the original mixture which comprises, contacting the mixture with a liqueed, normally gaseous parafnic hydrocarbon at a pressure sufliciently high to maintain the hydrocarbon in liquid phase and at a temperature suificiently elevated to insure the formation of two, separable immiscible liquid phases of different densities, the less dense phasecontaining a ratio of carotene to chlorophyll greater than that in the original mixture and the denser phase a ratio of chlorophyll to carotene greater than that in the original mixture, and separating the two phases.

8. A process of separating a crude chlorophyllcarotene-containing mixture into fractions one of which contains a greater ratio of carotene to chlorophyll than that in the original mixture and the other a ratio of chlorophyll to carotene greater than that in the original mixture which comprises, contacting said mixture with a liqueed, normally gaseous paraiiinic hydrocarbon in a continuous liquid-liquid extraction system at a pressure sufficiently high to maintain the hydrocarbon in the liquid phase and at a temperature sufficiently elevated to insure the formation of two immiscible liquid phases of different densities, continuously withdrawing from the system the less dense phase consisting primarily of the liquefied hydrocarbon in which the ratio of carotene to chlorophyll is greater than the ratio in the original mixture and continuousl ly withdrawing the denser liquid phase containing'a ratio of chlorophyll to carotene substantiallygreater than such ratio in the initial mixture.

9.` A process of separating an extract of plant tissue containing lipids, chlorophyll, carotene and tocopherols into two fractions one of which contains substantially all of the lipids, carotene and tocopherols and the other substantially all of the'chlorophyll which comprises, contacting such extract with a liquefied, normally gaseous para'inic hydrocarbon in a continuous liquidliquid extraction system at a pressure suiciently high .to maintain the hydrocarbon in the liquid phase land at a temperature sumciently elevated to insure the formation of two immiscible liquid phases of diierent densities, withdrawing from the system the lighter liquid phase containing substantially all the carotene, lipids and tocopherols with most of the hydrocarbon and a second liquid phase containing substantially all of the chlorophyll and the remainder of the hydrocarbon.

. ll0. A process of treating the crude extract of plant tissue containing chlorophyll and carotene to recover a carotene enriched and a chlorophyll enriched fraction which comprises, passing said extract to the upper portion of a continuous counter-current extraction zone, counter-currently passing a liquefied, normally gaseous parailnic hydrocarbon into the lower section of the zone, maintaining the hydrocarbon at a temperature sufficiently elevated to insure the formation of two immiscible liquid phases of different densities, continuously withdrawing from the upper section of the zone the less dense phase in which the carotene is largely concentrated, continuously withdrawing from the lower portion of the zone the denser liquid phase containing substantially all of the chlorophyll, removing the hydrocarbon from each phase to recover respectively a carotene enriched fraction and a chlorophyll enriched fraction and recycling the hydrocarbon separated from each of these to the said extraction zone.

11. A process in accordance with claim 5 comprising removing the hydrocarbon from the separated, less dense liquid phase to recover a carotene-enriched fraction.

12. A process in accordance with claim 5 comprising removing the hydrocarbon from the separated denserliquid phase to recover a concentrate enriched in chlorophyll.

v13. A process in accordance with claim 8 cornprising removing the hydrocarbon from the said less dense phase to recover a carotene-enriched fraction.

14. A process in accordance with claim 8 comprising removing the hydrocarbon from the denser liquid phase to recover a fraction enriched in chlorophyll.

RALPH MILLER.

REFERENQES errno The following references are of record in the file of this patent:

UNITED STATES PATENTS Number Name Date 2,118,454 Schaafsma May 24, 1938 2,131,394 Test Sept. 27, 1938 2,394,278 Wall et al. Feb. 5, 1946 2,394,968 Van Orden Feb. 12, 1946 

1. THE PROCESS OF FRACTIONATING A CRUDE CHLOROPHYLL-CAROTENE-CONTAINING MIXTURE INTO FRACTIONS ONE OF WHICH CONTAINS A GREATER RATIO OF CAROTENE TO CHLOROPHYLL THAN THAT IN THE ORIGINAL MIXTURE AND THE OTHER A RATIO OF CHLOROPHYLL TO CAROTENE THAT IS LARGER THAN THAT IN THE ORIGINAL MIXTURE WHICH COMPRISES, CONTACTING THE MIXTURE WITH LIQUID PROPANE AT A TEMPERATURE OF ABOUT 160*F. AND A PRESSURE SUFFICIENTLY HIGH TO MAINTAIN THE PROPANE IN THE LIQUID PHASE TO FORM TWO, SEPARABLE, IMMISCIBLE LIQUID PHASES, THE LESS DENSE PHASE CONTAINING A RATION OF CAROTENE TO CHLOROPHYLL GREATER THAN THE ORIGINAL MIXTURE AND THE DENSER PHASE A RATIO OF CHLOROPHYLL TO CAROTENE GREATER THANT THE ORIGINAL MIXTURE, AND SEPARATING THE TWO PHASES. 